U.S. patent number 4,152,242 [Application Number 05/425,089] was granted by the patent office on 1979-05-01 for immunodisc electrophoresis.
This patent grant is currently assigned to International Foundation of Microbiology. Invention is credited to Sanit Makonkawkeyoon.
United States Patent |
4,152,242 |
Makonkawkeyoon |
May 1, 1979 |
Immunodisc electrophoresis
Abstract
An electrophoretic gel apparatus and analytical method are
provided for analyzing samples without the need for removing the
gel from the apparatus. The electrophoretic gel is provided with a
central opening therethrough for accomodating an analytical
reagent, so that the latter may react with electrophoretically
separated components of a sample undergoing analysis.
Inventors: |
Makonkawkeyoon; Sanit
(Chiengmai, TH) |
Assignee: |
International Foundation of
Microbiology (Chicago, IL)
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Family
ID: |
22607078 |
Appl.
No.: |
05/425,089 |
Filed: |
December 17, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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167367 |
Jul 29, 1971 |
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Current U.S.
Class: |
204/606; 204/610;
422/82.01; 436/516 |
Current CPC
Class: |
G01N
27/44721 (20130101); G01N 27/44756 (20130101); G01N
27/44747 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 027/26 (); G01N
027/28 () |
Field of
Search: |
;204/18G,299,18R ;23/23B
;424/12 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Prescott; Arthur C.
Attorney, Agent or Firm: Parker; John L.
Parent Case Text
DESCRIPTION OF THE INVENTION
This is a continuation of my application Ser. No. 167,367 filed
July 29, 1971 now abandoned.
Claims
I claim as my invention:
1. Apparatus for carrying out analytical determinations on a sample
containing unknown components, comprising, in combination, a hollow
enclosure, an electrophoretic gel column within said enclosure and
adapted to receive the sample at one end thereof, means for
electrophoretically separating the various components of the sample
from each other and disposing them across the gel column at
intervals therealong, said gel column having a central bore therein
open for receiving an analytical reagent for contacting the
separated components of the sample disposed along the column.
2. A device for carrying out analytical determinations on a sample
containing unknown components, comprising, in combination, an
elongated container, an elongated electrophoretic gel disposed in
said container, and means for separating the components of the
sample into concentrated discs spaced along said gel, said gel
having a central bore therein open for receiving an analytical
reagent for contacting the separated components of the sample and
having an axis coincidental with that of said container.
3. Apparatus for use in electrophoresis and electrofocusing
including a hollow transparent elongated enclosure and an elongated
core member of smaller cross-section than the cross-section of the
enclosure within the enclosure, stopper means for closing one end
of said enclosure, said stopper means removably holding said member
in a selected position within said enclosure, and a separating gel
in said enclosure and between said core member and enclosure.
4. The structure of claim 3 wherein said enclosure is a tube and
said member is a rod.
5. The structure of claim 4 wherein said stopper means include
means for holding said rod coaxially with said tube.
6. The structure of claim 3 wherein said enclosure has an annular
cross-section.
Description
The present invention relates to the detection of antigens,
enzymes, and other biologically active and inactive components of
biological and other fluid and extracts using electrophoresis
techniques. More particularly, the invention is concerned with
improvements in disc electrophoresis techniques used in such
analytical determinations.
Disc electrophoresis using polyacrylamide and other suitable gels
has been used to characterize and resolve complex mixtures of
proteins such as serum, tissue extracts, and purified hormones and
enzymes. In this technique, a sample containing suspended particles
is placed on top of a column of polyacrylamide, starch or other
suitable gel capable of functioning as an electrolyte. Typically,
the column of gel is pre-polymerized in place in a glass tube or
like container. An electric potential is applied to electrodes in
contact with the suspension and with the bottom of the column, the
suspended particles become charged, and the various types of
particles move through the gel at different rates. The separated
components thus become concentrated in thin discs at spaced
intervals along the gel column. Upon discontinuance of the electric
potential, the gel column is removed from the glass tube or other
container, and the thin discs are physically separated from one
another, as by slicing. Then the individual disc portions are
individually reacted with analytical reagents as desired for
identification.
Such a technique has been generally described, for example, in U.S.
Pat. Nos. 3,384,564 to Ornstein et al., 3,445,360 to Via, and
3,576,727 to Evatt, as well as in Ann. N.Y. Acad. Sci., Vol. 121,
at p. 382 and at p. 428 (1964). Unfortunately, such prior disc
electrophoresis techniques have been unduly time consuming and
often result in at least partial loss of the samples, since the
discs must be cut from the gel column after the column has been
removed from the glass tube. Also, such prior techniques have not
been used to any great extent for determinations of antigenic
components of complex mixtures, mainly because the test reactions
taking place on a Petri dish outside of the gel column are
difficult to observe and read.
It is an object of the present invention to overcome these
disadvantages by providing an immunodisc electrophoresis analysis
technique for antigenic as well as non-antigenic components of
complex mixtures which eliminates the need to remove the gel from
the glass tube or other container, and thus avoids the possibility
that the gel column may become accidentally broken in an unwanted
manner. It is a related object of the invention to provide an
immunodisc electrophoresis analytical technique which not only
separates and concentrates the components of a test sample into
continuous thin discs within an electrophoretic gel column, but
which accomodates in situ reactions between the separated component
discs and appropriate analytical reagents (e.g. antisera) while the
discs are still in place within the column.
A further related object of the invention is to provide such an
immunodisc electrophoresis analytical technique in which all of the
test reactions may take place within the gel column, i.e. no longer
is it necessary to physically separate the component discs from the
column and from one another prior to carrying out further treatment
of the component discs with analytical reagents. An allied object
of the invention is to afford an analyst an analytical tool for
studying the antigenic properties of each of the disc components
separated in an immunodisc electrophoresis column by observing the
results of analytical reactions while the reactants remain inside
the column.
Yet another object of the invention is to provide a simple,
effective analytical disc electrophoresis technique which yields
sharply defined antigenic discs which are easy to count and may
readily be reacted in situ with antisera to provide positive sample
identification in minimum time.
Other objects and advantages of the present invention will become
apparent upon reading the following detailed description and upon
reference to the drawings, in which:
FIG. 1 is a perspective view of an illustrative device for
preparing an electrophoretic gel column which may be used in
carrying out the invention.
FIG. 2 is a perspective view similar to FIG. 1, showing an
electrophoretic gel column in place within the preparation device,
and a sample to be analyzed in place on top of the gel column.
FIG. 3 is a vertical, sectional schematic view of an illustrative
electrophoresis apparatus suitable for use in carrying out the
invention, showing the gel column in place within the
apparatus.
FIG. 4 is a perspective view of an illustrative larger
electrophoresis apparatus for handling a multiplicity of samples
simultaneously.
FIG. 5 is an enlarged, diagrammatic vertical cross-section of an
illustrative gel column after electrophoresis, showing the various
electrophoretically separated components of a sample (in stained
condition to render them readily visible).
FIGS. 6, 7 and 8 are enlarged vertical cross-section views of tubes
holding illustrative electrophoretic gel columns utilized for
different types of analyses carried out in accordance with the
invention. FIG. 6 is illustrative of an antigenic analysis, FIG. 7
depicts an enzymatic activity determination, and FIG. 8 is
illustrative of use of a developing agent, such as a dye, to stain
the electrophoresed components wherever they appear along the
column.
While the invention is described in connection with certain
preferred embodiments, it will be understood that I do not intend
to limit the invention to those embodiments. On the contrary, I
intend to cover all alternatives, modifications and equivalents as
may be included within the spirit and scope of the invention as
defined by the appended claims.
Turning now to the drawings, the illustrative electrophoretic
system includes a device 10 for preparing a gel column 11 (FIGS. 1
and 2), and a suitable electrophoresis apparatus 12, shown
schematically in FIG. 3, into which the gel column is placed for
electrophoresis. If desired, a multiplicity of electrophoresis
apparatuses 12 may be operated simultaneously within a larger
apparatus 14 (FIG. 4).
With particular reference to FIGS. 1 and 2, it is seen that the
illustrative gel preparation device 10 includes a vertically
disposed, elongated cylindrical sleeve or tube 15 mounted upon a
base 16, which as shown takes the form of a rubber stopper of a
type common to analytical laboratories. The rubber stopper 16
carries a cylindrical projection 16a sized to closely fit inside
the lower portion of the tube 15, so that a liquid tight seal
results when the lower end of the tube has been placed over the
stopper projection. The tube 15 is transparent to permit visual
inspection of its contents, glass or plastic tubing being preferred
materials of construction.
The gel column 11 is prepared by pouring or casting a suitable
separating gel, such as polyacrylamide, into the open upper end of
the tube 15 until the gel level nears the upper tube end (see FIG.
2). The gel quickly becomes a semi-solid, having pores of various
sizes for passing or retaining the different particle sizes present
in a given sample.
In carrying out the invention, means is provided for accomodating
analytical reactions between electrophoresed components of a sample
and selected analytical reagents in situ within the gel column. As
shown in the drawings, this is accomplished by forming the gel
column as a hollow cylinder, i.e. by providing an elongate central
opening or bore 18 passing longitudinally through the gel column
11, the opening providing a repository for analytical reagents
added to the column following electrophoresis. In this instance the
central opening 18 in the gel column 11 is formed by placing a
vertical elongated core rod 19 centrally within the tube 15 prior
to casting of the gel. The rod 19 is shown (FIG. 1) mounted upon
and supported by the stopper projection 16a, a suitable support
recess (not shown) being provided in the stopper for receiving the
lower end of the rod. The rod 19 -- stopper 16a connection may be
screw-threaded or simply a friction fit. Preferably the rod 19
extends beyond the top of the tube 15 to permit ready manual
removal of the rod later (see below).
Thus, in preparing the electrophoretic gel column 11, the gel is
poured into the tube 15 around the core rod 19 in such a manner
that it surrounds the rod. The gel is then allowed to set
(polymerize) with the rod in place, so that a cylinder of gel is
formed having the vertical rod in its center (see FIG. 2). The gel
column 11, tube 15 and rod 19 combination is then separated from
the rubber stopper 16 and placed in the electrophoresis apparatus
12. A sample containing antigenic, enzymatic or other components to
be analytically determined may then be poured onto the top of the
gel column 11, to form a sample layer 20 thereon.
Any of various electrophoretic gels may be used with the invention,
for example polyacrylamide, agar, starch, pectin, sephadex,
cellulose, or any other substance which in aqueous or organic
fluids forms a clear, transparent, translucent, or colored gel and
which is capable of being packed to a semi-solid condition to form
a gel cylinder. The gel cylinder may be cast in tubes of various
lengths and diameters as desired. The casting can be carried out
while the tube 15 is in a vertical or horizontal position, either
manually, using pressure, or by vacuum operated casting devices.
While the base 16 for the gel preparation device is shown as a
rubber stopper, other base materials such as a screw cap or plastic
closure may be used as well. The core rod 19 may be formed of
glass, plastic or metal, should have a diameter smaller than the
inside diameter of the tube 15, and should be of sufficient length
to facilitate its later manual removal from the tube.
As will be seen from FIG. 3, the electrophoresis is carried out
with the rod 19 in place within the tube 15. The electrophoresis
apparatus 12 includes a suitable support structure (not shown) for
mounting the gel tube 15, column 11 and embedded rod 19 between
upper and lower reservoirs 21, 22, respectively. A source of
electric current 24 is provided, suitably connected to positive and
negative electrodes 25, 26 disposed in the upper and lower
reservoirs, respectively, and the reservoirs are filled with
appropriate buffer solutions 28, 29 of a type well known to those
skilled in the art.
Upon application of electric potential to the electrodes 25, 26,
electrophoresis takes place, with the suspended particles in the
sample layer 20 becoming electrically charged and moving downwardly
through the gel column 11 at different rates of travel depending
upon their nature. The gel containing tube 15 is left in this
position in the electrophoresis apparatus 12 for whatever length of
time is necessary for the components of the sample to migrate from
the top of the gel column 11 downwardly to various longitudinally
spaced positions within the gel. After the desired time of
electrophoresis has elapsed, the tube 15, gel column 11 and rod 19
are disengaged from the source of electric current and the central
rod 19 is removed manually from the gel column. At this point the
gel column 11, which is hollow by reason of its central bore or
lumen 18, has the annular or hollow cylinder appearance depicted in
FIG. 5. The sample components have been completely separated from
each other along the column, and appear as a plurality of
individual discs 30.
In the event that the larger electrophoresis apparatus of FIG. 4 is
used for simultaneous preparation of multiple samples, the
operation is like that described above as concerns FIG. 3. Thus,
the larger apparatus 14 includes upper and lower reservoirs
containing buffer solutions 28a, 29a into which the ends of a
plurality of gel tubes 15a are immersed. The gel tubes 15a include
central rods 19a, with sample layers 20a being present at the
beginning of electrophoresis. Electric current is supplied to the
larger electrophoresis apparatus through electrodes 25a, 26a.
Following electrophoresis to separate the components of the sample
into discs spaced along the gel column, the bore or lumen 18 of the
column may be filled with any desired analytical reagent, and the
column incubated, whereupon localized test reactions may be caused
to take place at the locale of each disc. For example, analytical
reagents used may include antisera, stain or a suitable substrate,
or combinations thereof, depending upon the nature of the
analytical determination being carried out. Importantly, the
analytical procedure avoids the necessity for removing the gel
column 11 from the tube 15.
FIGS. 6, 7 and 8 depict several ways in which the present invention
may be utilized in conducting analytical determinations. The
illustrative of FIG. 6 relates to determination of the antigenic
components of a sample. Following electrophoresis of the separate
antigenic components into discs spaced along the gel column 11, the
tube 15 and gel column are removed from the electrophoresis
apparatus and placed in a horizontal position. Then the bore or
lumen 18 of the gel cylinder is filled with antiserum containing
antibodies to all or some of the antigenic components of the
sample. Alternatively, the antiserum may be premixed with a gelling
agent, such as agar, so that after being placed in the cylinder it
forms an inner gel and serves as a repository for the antiserum.
The tube 15 is then suitably sealed at both ends to prevent
evaporation, e.g. with plastic film, and the antigen-antibody
reaction is allowed to take place by incubating the tube at the
desired temperature. The antigen-antibody reaction results in
precipitation which appears as white bands 31 either in the outer
gel column or in the inner gel, and the test results are read as
such.
FIG. 7 depicts the results of a determination of the enzymatic
components of a sample. Following electrophoretic separation of the
individual enzymatic components into discs spaced along the column
11, the cylinder bore 18 is filled with the substrate of the enzyme
whose activity, location, and number is being analyzed. The
substrate may be premixed with any necessary indicator system for
detecting enzymatic activity, or the substrate and indicator system
may be mixed with a gelling agent before being placed in the bore.
The gel cylinder filled with these agents is then incubated at the
desired temperature and the location of the particular enzymes
noted from the presence of pockets 32 of precipitate.
Alternatively, the hollow gel cylinder 11 may be filled with a
developing agent, such as a dye, which will stain the sample
components wherever they may be located in the gel. In this event,
the cylinder bore 18 is filled with a stain or other developing
agent, and the agent is allowed to remain within the bore until the
components of the sample have been stained or have otherwise become
altered so that they may be observed (see the bands 34 of FIG. 8).
The excess stain or the agent is then removed by filling the
cylinder bore with washing solution of a composition compatible
with the stain or other reagent used.
It will thus be seen that the invention may be utilized in the
analysis of antigenic as well as non-antigenic components of
biological and other fluids and extracts. For example, the
improvements described herein may be used in the analysis of
enzymatic, hemolytic, and toxic components of such fluids and
extracts. Moreover, the advantages and objects of the invention may
be attained in various ways. By utilizing the invention, an analyst
may study the enzymatic properties of electrophoretically separated
sample components by reacting them with an appropriate substrate
disposed in the hollow portion of the gel column. The
enzyme-substrate reactions may be visualized, if necessary, through
use of indicators present in the substrate or applied separately
after the enzymatic reactions have altered the substrate. The
invention advantageously also may be used in studying different
forms of isoenzymes, either by serological or biochemical reactions
taking place within the gel column.
* * * * *